PB2174: DISSECTING THE MECHANISM OF INTERFERON ALFA (IFNΑ) RESPONSE IN MYELOPROLIFERATIVE NEOPLASM AND IDENTIFICATION OF PREDICTORS OF RESPONSE
Authors: Eli Soyfer, Jianhong Heidmann, Angela Fleischman
Journal: HemaSphere (2023)

Topic: 15. Myeloproliferative neoplasms - Biology & Translational Research Background: Myeloproliferative Neoplasm (MPN) is a group of chronic blood cancers characterized by the somatic acquisition of mutations, in particular Janus Activated Kinase 2 (JAK2V617F), which lead to constitutive activation of myeloid cytokine receptors. Interferon alfa (IFNα) is the only known therapy that induces a molecular response by decreasing the Variant Allele Frequency (VAF) of JAK2V617F. However, not all MPN patients on IFNα enjoy a molecular response, and why some patients respond, and others do not remains unclear. Our long-term goal is to identify early biomarkers that predict for a molecular response to IFNα by following in depth MPN patients who are beginning IFNα dosing for one year with daily sampling of leukocyte DNA to monitor day-to-day changes in the driver mutation VAF using Digital PCR (dPCR). Aims: The goal of this project is to develop strategies to use non-invasive sampling sources elucidate what factors are responsible for this disparity in IFNα effectiveness, so to allow for more detailed clinical guidance in early IFNα dosing that can maximize response over resistance. We will collect daily saliva samples for one year from MPN patients initiating IFNα therapy. This will allow us to intensely follow the daily dynamics of the JAK2V617F allele burden in response to IFNα. As the success of this aim is dependent upon patient participation another critical component of this Aim is optimization of participant compliance, engagement, and satisfaction. Methods: Saliva collection: 0.1% gelatin+PBS rinse for 30 sec, repeated three times, then vortexed for 1 min, and centrifuged at 500 RCF/10 min/4°C. The pellet was resuspended in 2 mL PBS, filtered, and centrifuged again. The pellet was resuspended in 0.5% BSA+PBS for flow cytometry, or 0.1% FBS+PBS for histology. dPCR: DNA was added to master mix, JAK2WT and JAK2V617F specific primers and fluorescent probes, and sterile water. dPCR protocol: 10-minute pre-heat at 96°C, followed by 45 cycles of denature at 96°C for 30 seconds/anneal-extend at 61°C for 60 seconds, analyzing for JAK2WT and JAK2V617F fluorescent signals to quantify the amount of DNA (cp/µL) per allele. VAF was calculated using the amounts of DNA (JAK2V617F)/(JAK2WT+JAK2V617F). Results: We first demonstrated that saliva can serve as a viable alternative to blood for isolation of leukocyte DNA. We confirmed that saliva contains abundant leukocytes in both healthy controls as well as patients with hematologic malignancies, via histology and flow cytometry. We then used dPCR to compare the interchangeability of blood and saliva for somatic mutation detection in a set of 35 JAK2V617F -positive MPN. We found that concurrently collected blood and saliva were virtually interchangeable for the detection of JAK2V617F, even at VAF’s < 1%. Using stored samples from patients received before and during IFNα treatment, we see variable responses of the JAK2V617F VAF to IFNα. We have recruited three patients so far to intensely monitor JAK2V617F VAF early on during IFNα treatment. Our first patient has completed their first four months of IFNα use and so far, we can see a slight general uptrend of VAFs. Interestingly, we observed a 2cm increase in length over baseline in the first month of treatment too. Summary/Conclusion: Here, we explore the novel use of saliva for comprehensive VAF tracking of MPN patients in a remote setting. We have demonstrated that saliva is an equivalent source to peripheral blood to quantify JAK2V617F VAF with dPCR. We will continue to follow the day-to-day variation of VAF in response to IFNα and plan to expand the use of saliva to gene expression profiling. Overall, the results of this trial will be used to generate a preliminary model of IFNα response, which can be used to predict how likely a MPN patient will benefit from IFNα therapy. Keywords: Pegylated Interferon, Myeloproliferative disorder